Cellulose is relied upon as the raw material for a number of useful products including paper products and wound dressings. Cellulose may be obtained from plants and various microorganisms in culture, for example from the cellulose producing bacteria of the genus Acetobacter. Acetobacter is characteristically a Gram-negative, rod-shaped bacterium 0.6-0.8 um by 1.0-4 um. It is strictly aerobic; metabolism is respiratory, never fermentative. It is further distinguished by the ability to produce multiple poly .beta.(1-4)-glucan chains, chemically identical to cellulose. Multiple cellulose chains or microfibrils are synthesized at the bacterial surface at sites on the cell wall. The production of cellulose by Acetobacter has been the subject of intense study since at least the 1930's. In particular, Acetobacter xylinum has been widely studied to attempt to elucidate the mechanism of cellulose synthesis in intact cells [Schramm and Hestrin,(1954) J. Gen. Microbiol. 11:123-129].
The enzymatic pathway for cellulose synthesis in Acetobacter xylinum has been investigated and essentially four enzymatic steps have been characterized in cell free extracts of A. xylinum which appear to comprise the complete pathway from glucose to cellulose. These are the phosphorylation of glucose by glucokinase [Benziman, et al., (1972) J. Bacteriol., 111:325-330], the isomerization of glucose-6-phosphate to glucose 1-phosphate by phosphoglucomutase [Gromet. et al.. (1957) Biochem. J., 67:679-689; Frei-Roitman, Factors affecting the activity of phosphoglucomutase and UDP-glucose pyrophosphorylase of Acetobacter xylinum, M.Sc. thesis, The Hebrew University of Jerusalem, Jerusalem, Israel (1974)]; the synthesis of uridine 5'-diphosphoglucose (UDP-glc) by UDPG-pyrophosphorylase, [Frei Roitman, supra; Swissa, Biosynthesis of cellulose in Acetobacter xylinum, Ph.D. thesis, The Hebrew University of Jerusalem, Jerusalem, Israel (1978)], and the cellulose synthase reaction.
Attempts to purify cellulose synthase from a strain of A. xylinum employing conventional chromatographic techniques have not been especially successful, but recently the enzyme has been significantly purified (P. Ross and M. Benziman (1989) in Biosynthesis and Biodegradation of Cellulose and Cellulose Materials, eds. Weimar and Higler, Marcel Dekker, Inc. NY), and its properties and structure in the purified state are currently under investigation.
Similarly, attempts to purify cellulose synthase by in vitro cellulose entrapment and chromatographic techniques have resulted in a partially purified 83 kilodalton (kd) polypeptide (Lin and Brown, The Tenth Cellulose Conference, May 29-Jun. 2, 1988, Abstract BGl, page 27).
Although the physiological role of cellulose synthesis in this organism is still not clear, considering that at least 10% of the cell's energy budget is devoted to cellulose production at any one time [Weinhouse, Regulation of Carbohydrate metabolism in Acetobacter xylinum, Ph.D. thesis, The Hebrew University of Jerusalem, Jerusalem, Israel (1977)], it is not surprising that the biosynthetic system is governed by a complex regulatory system.
Cellulose synthase, the only enzyme unique to the pathway, performs the "committed" step in cellulose formation--a metabolic dead-end with regard to carbon utilization--and hence would logically be the prime candidate for strict regulatory control. Furthermore, as demonstrated in cell-free extracts, the level of enzyme activities leading to UDP-glc are in large excess relative to that of the cellulose synthase, strongly supporting the proposition that the latter comprises the rate limiting step in cellulose biosynthesis.
A more complete knowledge of the biochemistry of cellulose synthesis would facilitate greater productivity and yield of cellulose from cultures of cellulose producing microorganisms. The growth of bacterial cells in culture is observed to be initially exponential but slows as the cells enter a stationary growth phase. The majority of cellulose is produced later in fermentation when the number of cells is highest, however the amount of cellulose made per cell per unit time (specific productivity) declines as the fermentation proceeds. It is believed that cellulose synthase activity may be rate limiting as cells in culture reach the stationary growth phase. One improvement in cellulose production would be to remove a rate limiting step in cellulose synthesis, thereby preventing the observed decline in cellulose specific productivity in culture.
Recombinant DNA techniques are now routinely available for production of desired proteins. However, to take advantage of such recombinant DNA techniques, the gene coding for the desired protein, such as cellulose synthase, must first be isolated. This task is considerable, especially when the primary sequence of the encoded protein is unknown and known assays for determining cellulose synthase activity are difficult.
The ability to produce recombinant cellulose synthase provides an important tool useful in exploring the mechanisms of cellulose synthesis, ultimately providing enhanced cellulose production from bacterial culture.